| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Pharmacology and Physiology and the Cancer Center University of Rochester School of Medicine and Dentistry Rochester, New York 14642
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
q/11
and Gß
can activate PLCß3, whereas only Gq/11 activates
PLCß1 effectively. In knockout mice, PLCß3 was absent, PLCß1 was
not up-regulated, and PLCß2 and PLCß4 were not expressed. Since
somatostatin inhibited influx of extracellular calcium in pituitary
cells from wild-type and PLCß3 knockout mice, the somatostatin signal
pathway was intact. However, somatostatin failed to increase
intracellular calcium in pituitary cells from either wild-type or
knockout mice under a variety of conditions, indicating that it did not
stimulate PLCß3. In contrast, somatostatin increased intracellular
calcium in aortic smooth muscle cells from wild-type mice, although it
evoked no calcium response in cells from PLCß3 knockout animals.
These results show that somatostatin, like other Gi/Go-linked hormones,
can stimulate a calcium transient by activating PLCß3 through
Gß, but this response does not normally occur in pituitary cells.
The densities of Gi and Go, as well as the relative concentrations of
PLCß1 and PLCß3, were similar in cells that responded to
somatostatin with an increase in calcium and pituitary cells. Calcium
responses to 1 nM and 1
µM TRH and GnRH were identical in pituitary
cells from wild-type and PLCß3 knockout mice, as were responses to
other Gq-linked agonists. These results show that in pituitary cells,
PLCß1 is sufficient to transmit signals from Gq-coupled hormones,
whereas PLCß3 is required for the calcium-mobilizing actions of
somatostatin observed in smooth muscle cells. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-subunits of G proteins in the Gq family. PLCß1 and ß3 show high
sensitivity to Gq (3, 4, 5, 6). PLCß2 and PLCß3 are also stimulated by
G protein ß-subunits (1, 7, 8, 9, 10, 11). In particular, ß-subunits
derived from the pertussis toxin-sensitive Gi and Go proteins have been
implicated in activation of PLCß2 and PLCß3, accounting for the
pertussis toxin-sensitive calcium transients sometimes seen when
Gi/Go-linked receptors are activated, most notably in hematopoietic
cells but also in neuronal, smooth muscle, and fibroblast cells.
In vitro, Gq stimulates PLC- mediated
phosphatidylinositol (4, 5) bisphosphate hydrolysis at much lower
concentrations than Gß (3, 11, 12).
In the anterior pituitary gland, TRH and GnRH are coupled to Gq and
stimulate secretion, whereas somatostatin and dopamine are coupled to
Gi/Go and inhibit secretion. TRH and GnRH produce strong calcium
transients, initially by releasing intracellular calcium and
subsequently by stimulating calcium influx (13, 14, 15). The role of
different PLCß isoforms in these responses has not been clarified.
The somatostatin and dopamine D2 receptors serve to inhibit release of
hormones and reduce levels of hormone transcription in the
pituitary gland, where they limit calcium influx (16, 17, 18, 19, 20). These
various effects are achieved through either GTP-activated forms of
Gi or Go or through ß-subunits from Gi/o proteins.
Interestingly, somatostatin causes a calcium increase in certain
nonpituitary tissues such as intestinal smooth muscle (21), apparently
by activating PLCß3.
The balance of activating and inhibitory hypothalamic hormones is critical for normal pituitary function. It is not known which PLCß enzymes are expressed in pituitary, what role the different isoforms play, and why Gi/Go-coupled receptor systems produce different calcium responses in different cell types. We have taken advantage of mice with targeted disruption of the PLCß3 gene to determine the importance of this isoform in calcium responses to TRH and GnRH, which act on Gq-coupled receptors, and somatostatin, which acts via Gi/Go.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Somatostatin works through Gi/Go and in principle might stimulate
PLCß3 by releasing Gß. The peptide has been reported to mobilize
intracellular calcium in smooth muscle cells (23, 24). We determined
the ability of somatostatin to increase intracellular free calcium in
aortic smooth muscle and anterior pituitary cells from PLCß3-knockout
and wild-type animals. We also tested a mixture of agonists for
Gq-linked receptors commonly expressed on cells (endothelin, bombesin,
and bradykinin); these agonists (Gq mix) are expected to stimulate any
PLCß by activating Gq. The combined Gq agonists were applied at
the end of each experiment, and only those cells that responded to them
with a calcium increase were considered potentially responsive to
somatostatin, since these cells were viable with an IP3-releasable
calcium pool.
Calcium Responses to Somatostatin in Aortic Smooth Muscle Cells
from PLCß3+/+ and PLCß3-/- Mice
Cells prepared from the thoracic aorta of mice were stained with
antibody against smooth muscle actin to verify cell phenotype (Fig. 1C
). For measurement of intracellular
free calcium, cells were loaded with fura2. The calcium responses of
aortic smooth muscle cells from PLCß3+/+ and PLCß3-/- animals to
agonists for Gq-coupled receptors were not significantly different,
with 59 and 63% of cells responding with peak increases in 340/380
fluorescence ratios of 4.87 ± 0.05 and 5.03 ± 0.08,
respectively (Fig. 1, A and D). In contrast, somatostatin caused an
increase in calcium in aortic smooth muscle cells from wild-type but
not knockout mice. In PLCß3+/+ mice, somatostatin increased calcium
in 45% of responsive aortic smooth muscle cells, and the average peak
height was 2.77± 0.08 times baseline (Fig. 1, B and D). Somatostatin
mobilized calcium only at concentrations of 100 nM or
greater. In contrast, no cells from PLCß3-/- mice (of 27 Gq-agonist
responsive cells in 5 experiments) responded to somatostatin with any
increase at all in intracellular calcium. These results indicate that
the calcium response to somatostatin seen in smooth muscle cells
requires PLCß3.
|
). The somatostatin response in HEK293
cells was observed in essentially all cells and was completely
eliminated by pertussis toxin (Fig. 2B). All cells responded to Gq mix,
but pertussis toxin had no effect on this response.
|
, A and B). Again,
somatostatin never caused an increase in cytoplasmic calcium. To show
that somatostatin receptors were functional, we added somatostatin
after giving a mix of Gq-linked agonists (endothelin, bombesin, and
bradykinin) or depolarizing with high potassium. In these paradigms,
somatostatin decreased intracellular calcium in cells from both
PLCß3+/+ and PLCß3-/- mice (Fig. 3, C and D). This indicates that
the somatostatin signaling pathway, from receptor to G protein, was
functioning effectively in pituitary cells from wild-type and knockout
mice. The results also suggest that voltage-gated calcium entry
accounts for the sustained calcium rise after activation of Gq-linked
receptors.
|
PLC Isoforms in PLCß3+/+ and PLCß3-/- Mice and HEK293
Cells
To determine which isoforms of PLCß are expressed in mouse
pituitary cells, we performed Western blotting using pituitary tissue
obtained from wild-type and PLCß3 knockout animals (Fig. 4). Samples from wild-type (+/+) and
mutant (-/-) cell homogenates were loaded in equivalent amounts in
each gel and blotted with antibodies against PLCß1, -2, -3, or -4.
The specificity of antibodies to PLCß1, -2, and -3 was verified using
purified PLCs and blocking peptides. In blots for PLCß4, we used
cerebellar protein from wild-type and PLCß4 knockout animals (28) for
positive and negative controls, respectively (Fig. 4D, lanes a and b);
cerebellum is rich in PLCß4 (29). Anterior pituitary cells from
either the wild-type or PLCß3-knockout mice expressed no detectable
PLCß2 (Fig. 4B) or PLCß4 (Fig. 4D). As expected, PLCß3 was absent
from pituitaries of knockout animals (Fig. 4C). There was,
however, a significant amount of this isoform in the pituitaries
of wild-type mice, as evidenced by the dark band. PLCß1 was present
in pituitaries from PLCß3+/+ and PLCß3-/- mice (Fig. 4A). In
three separate experiments, the intensity of the PLCß1 bands appeared
the same for pituitary tissue from wild-type and knockout animals,
indicating that PLCß1 is not generally up-regulated to compensate for
the lack of PLCß3.
|
). As
shown in Fig. 5, HEK293 cells express
PLCß1 (panel A) and PLCß3 (panel B) but not PLCß2 (panel E).
Immunoblotting was performed under the same conditions for pituitary
and HEK293 cells. The ratio of PLCß3 to PLCß1 was similar in HEK293
cells, which respond to somatostatin with an increase in calcium, and
pituitary cells, which do not.
|
). The amount of Gi staining was
similar in both cell types. The antibody used recognizes isoforms 1, 2,
and 3, which did not separate under the conditions of electrophoresis;
the pattern shown in Fig. 6 is typical for mouse Gi (30). The amount
of Go appeared to be somewhat lower in aortic smooth muscle than in
pituitary cells. Go is reported to run as three major bands
representing the two splice variants of the protein and a
posttranslationally modified form (31). These results suggest that the
differences seen between pituitary and other cell lines are based on
something more complex than the total G protein complement
available.
|
q/11 to activate phosphatidylinositol turnover (15, 32, 33, 34, 35).
Typical calcium responses of pituitary cells from PLCß3+/+ and
PLCß3-/- mice to high doses of TRH, GnRH, or the Gq mix (50
nM bombesin, 50 nM bradykinin, 10
nM endothelin) are shown in Fig. 7. The responses of cells from intact and
PLCß3 knockout mice appeared very similar. Upon agonist addition,
there was a rapid increase in intracellular calcium due to release from
intracellular stores, followed by a slow decline over a period of
50–200 sec. The proportion of cells responding and mean peak height
attained after application of these agonists are shown in Fig. 8. Although the average responses to GnRH
and TRH were comparable in wild-type and knockout pituitary cells,
there was significant variability between individual cells within
each experiment. Some cells responded robustly immediately, others
exhibited a lag phase after agonist addition before responding, and
some did not respond at all. We were not able to carry out an extensive
analysis of the effect of sex and age on calcium responses, but in
general, female pituitaries had slightly fewer TRH-responsive cells
than their male counterparts (12–17% vs. 20%) (Table 1). Male and female pituitary cells gave
comparable peak responses to 1 µM GnRH or TRH.
There were no significant differences between mutant and wild-type
pituitary cells in terms of percentages of responsive cells or the
average maximal peak height for either sex (Table 1).
|
|
|
, D and H). The calcium mobilization patterns
looked very similar to those resulting from higher concentrations of
agonists or from the Gq cocktail. The lag time between addition of
agonist and an increase in calcium was often longer at lower agonist
doses, but the characteristic two-phase response was produced. Of note
is that, again, the general shape of the responses was similar for
wild-type and PLCß3-deficient cells. Between 35% (wild-type) and
38% (knockout) of cells responded to 1 nM GnRH. The
percentage of responsive cells was somewhat higher with 1
nM than 1 µM GnRH (Fig. 8). This may be a
reflection of the age and sex of the animals used, since lower-dose
experiments were done with older males. In the case of 1 nM
TRH, 16% (wild-type) to 21% (PLCß3-knockout) of cells responded
with increases in intracellular calcium. When we reduced the
concentration of GnRH and TRH to 0.1 nM, the fraction of
responsive cells fell to approximately 5%, precluding quantitative
analysis, but calcium responses were still observed in pituitary cells
from the knockout mice.
Gq Staining in Pituitary Cells from PLCß3+/+ and PLCß3-/-
Mice
Since Gq is expected to stimulate both PLCß1 and PLCß3, our
results raise the question of how pituitary responses to Gq activation
remained normal in PLCß3 knockout mice. As shown above in Fig. 4, PLCß1 was not up-regulated in pituitaries from knockout animals to
compensate for the lack of PLCß3. Another possible means of
compensation for the absence of PLCß3 is at the level of the G
protein. Immunoblotting of protein samples from pituitaries for the
common region of the -subunits of Gq and G11 was unsuccessful
because of the low abundance of this subunit. Immunofluorescent
staining for Gq/11 -subunit in pituitary cells from wild-type and
mutant animals revealed no discernible difference in staining of cells
from wild-type and knockout mice; both showed a largely membrane-bound
distribution of Gq/11 (Fig. 9). The
intensity of staining, indicative of the amount of protein present, was
also comparable in both samples.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
- and ß-subunits
of Gi/o proteins, acting either individually or in concert, are
central. In smooth muscle cells, somatostatin can act as a calcium-mobilizing hormone (23, 27, 42). Somatostatin has been reported both to increase phosphoinositide turnover and intracellular calcium and stimulate contraction in various smooth muscle cell preparations (21, 24), although it has also been found to inhibit contraction (43).
Several lines of evidence indicate that in nonhematopoietic cells,
Gß stimulation of PLCß3 is responsible for IP3 production and
calcium mobilization by Gi/Go-coupled receptors, including somatostatin
receptors. For example, overexpression of transducin , which binds
Gß, inhibits calcium responses via pertussis toxin-sensitive
receptors (9), as does antibody to PLCß3 and Gß (23). Our data
showing that somatostatin does not increase calcium in cells from
PLCß3-knockout mice provide additional strong support for this model.
Together with the finding that calcium responses via the µ-opioid
receptor are absent in the PLCß3-/- mice (22), these results can be
extrapolated to suggest that pertussis toxin-sensitive calcium
responses in nonhematopoietic cells in general are caused by Gß
activation of PLCß3.
It is less clear why somatostatin fails to initiate a calcium transient
in cell types like pituitary, where somatostatin evokes other calcium
responses. Differences in receptor subtypes or density, G protein
repertoire, or effector abundance all have the potential to affect the
coupling of the receptor. Mouse pituitary and aortic smooth muscle
cells were not attainable in sufficient quantities to permit analysis
of somatostatin receptor subtype expression, but all somatostatin
receptor subtypes are expressed in pituitary glands of other species
and the subtypes most effective in activating PLCß3, SSTR2 and 5, are
present at high levels in somatotrophs (21, 39, 40, 44). Pituitary
cells from wild-type and PLCß3-/- mice showed normal inhibitory
responses to somatostatin, indicating that they had an intact
somatostatin receptor-G protein pathway. We also showed that pituitary
cells express the G protein partners of the somatostatin receptor, Gi
and Go, at densities at least equivalent to those in aortic smooth
muscle cells, although we did not rule out possible differences in
Gi subunits. Finally, we showed that the requisite effector is
present, because wild-type mouse pituitary contained PLCß3 in
quantities equivalent to those in responsive fibroblasts, although the
distribution in different pituitary cell types is not known. One
potential explanation for the failure of pituitary cells to show a
calcium response to somatostatin is that alternative effectors such as
calcium channels engage available Gß. Another is that pituitary
somatostatin receptors are spatially organized in a manner that favors
regulation of ion channels and adenylyl cyclase but minimizes
activation of PLCß3. Since some somatostatin receptor subtypes have
PDZ domain-binding motifs at their carboxyl termini, they may interact
with scaffolding proteins that could be expressed in a cell
type-specific pattern. A number of proteins with PDZ domains have
recently been shown to interact with the type 2 somatostatin receptor
(45, 46). It is not obvious how this would account for activation of
PLCß3 in some cells but not others, because PLCß3, according to
fluorescent microscopy, appears to be distributed throughout the
cytoplasm (data not shown). Furthermore, calcium mobilization through
the type 2 receptor reportedly does not require the C terminus of the
receptor (41).
TRH and GnRH both exert their primary effects via Gq/11-mediated activation of PLCß. To our knowledge, single cell responses of mouse pituitary cells have not been described previously. Not surprisingly, the characteristics of the responses were quite similar to those measured in rat pituitary glands under very similar conditions (47). Our findings that TRH and GnRH responses are unaffected by disruption of the PLCß3 gene imply that G protein activation of PLCß1 is sufficient to support a full calcium response. The fact that PLCß3 knockout mice develop normally, are normal size, and are fertile is consistent with our finding of normal TRH and GnRH responsivity. The only phenotype known to result from disruption of the PLCß3 gene is exaggerated sensitivity to morphine analgesia (22) and formation of skin ulcers on the neck and behind the ears (48). It appears that the ability of Gi/Go-linked receptors to increase calcium in smooth muscle is nonessential. We have shown that the pituitary gland normally expresses just two G-protein- regulated PLCs, ß1 and ß3. Mice with targeted disruption of the PLCß1 gene have been described and are reported to live for only a few weeks (49). Their growth is severely retarded, consistent with a critical role of PLCß1 in endocrine function. The mice also suffer from severe seizure disorders.
A major question raised by our data is why elimination of PLCß3,
which is strongly activated by the -subunit of Gq/11, had no effect
on calcium responses to Gq-activating hormones. We considered the
possibility that high doses of TRH and GnRH increased IP3 to levels
above those necessary for a maximal calcium response, masking possible
differences. However, we were unable to detect differences between
wild-type and knockout animals when we tested hormones at
concentrations so low that only rare cells responded. We ruled out the
possibility that PLCß1 was up-regulated in pituitary as a whole,
although we did not determine its distribution in individual cells, and
we ruled out the possibility that a PLCß isoform not normally
expressed in pituitary was turned on in the PLCß3-/- mice. Although
immunocytochemistry is not quantitative, we also saw no difference in
staining for the common region of the - subunits of Gq and G11,
the relevant G proteins. Taken together, these findings suggest that
cells do not compensate for the lack of PLCß3 by increasing the
concentration of receptors, G proteins, or PLCß1. There may be
another mechanism for compensation for the lack of PLCß3.
Alternatively, TRH and GnRH may normally stimulate PLCß1 but not
ß3; if so, the basis for such specificity is unknown. The activity of
PLCß3 can be inhibited by a cAMP-dependent phosphorylation (50), and
it could be that the levels of cAMP in the pituitary are sufficient to
render PLCß3 largely inactive under normal conditions. The PLC
enzymes may be functionally compartmentalized, or there may be
accessory proteins that promote Gq activation of PLCß1 or prevent
activation of PLCß3.
Our studies with PLCß3 knockout mice provide compelling evidence that
stimulation of PLCß3 by Gß leads to the release of intracellular
calcium in those tissues where activation Gi/Go-coupled receptors
provoke an increase in calcium. PLCß3 is abundant in anterior
pituitary, even though agonists for Gi/Go- coupled receptors do not
release intracellular calcium in intact pituitary cells. Calcium
responses to TRH and GnRH appeared to be intact in pituitaries from
PLCß3-/- mice, showing that PLCß1 is adequate for normal signal
transduction.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
i and o
subunits, and blocking peptides were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Purified antiserum to
Gq/11 was a generous gift from Dr. Paul Sternweis (University of
Texas, Dallas, TX). Rhodamine- and fluorescein-conjugated secondary
antibodies were from American Qualex (LaMiranda, CA).
Calcium Measurement
Measurements of intracellular calcium were performed at 37 C
essentially as described by Nelson and Hinkle (51). Cells were loaded
at room temperature with 4 µM Fura2-AM, 0.2% BSA, and 20
µg/ml cyclosporin A for 40–50 min in the dark in HBSS buffered with
15 mM HEPES to pH 7.4, rinsed, and placed in a Sykes-Moore
chamber from Bellco (Vineland, NJ) covered with 1 ml HBSS. Single-cell
calcium experiments and imaging were performed on a Nikon
inverted microscope with a DAGE CCD72 camera and Geniisys intensifier
system (Michigan City, IN) using Image-1 or Metafluor software
from Universal Imaging Corp. (Media, PA). Results show traces from
individual cells within a field. All traces depict cells that were
responsive to a mixture of Gq-activating agonists, and all experiments
were repeated at least three times with comparable results.
Pituitary and aortic smooth muscle cells were obtained from 6- to 12-week-old mice after cervical dislocation or CO2 asphyxiation. Between two and four animals were used for each preparation. Pituitary glands were transferred under aseptic conditions to 100 µl of 1.25 mg/ml trypsin in EDTA and chopped finely with a razor blade. The tissue was then enzymatically dispersed by two 5-min and one 10-min incubation in 1 ml trypsin at 37 C, followed by one 20-min incubation in 1 mg/ml type I collagenase in DMEM. Cells were dispersed mechanically with a pipette tip, resuspended in growth medium [DMEM supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), fungizone (1 µg/ml), and kanamycin (100 µM)], and plated on Cell-Tak-coated glass coverslips in growth medium and grown 24–48 h before use. For calcium experiments, cloning rings were used to confine the cells in a small area.
To isolate aortic smooth muscle cells, a 1-cm length of thoracic aortic tissue was removed and rinsed briefly in sterile HBSS. The tissue was then transferred to 1 ml of sterile HBSS containing 1.3 mg/ml type I collagenase and 0.3 U/ml type I elastase and incubated 40 min at 37 C. Adventitia were removed and the tissue placed in 1 ml sterile HBSS with 2 mg/ml collagenase and 3.3 U/ml elastase and minced using a sterile razor blade. After a 60-min incubation at 37 C, a final enzymatic digestion in 1 ml of 3 mg/ml collagenase for 20 min at 37 C was performed. Cells and remaining tissue fragments were mechanically dispersed, and the cells were collected by centrifugation, resuspended in growth medium, and plated on coverslips coated with poly-L-lysine. Cells were grown at 37 C in a humidified atmosphere and the medium was changed daily until they had reached near confluence, within 4–7 days. Cell type was verified by immunofluorescent staining for smooth muscle-specific actin.
Protein Isolation and Western Blotting
For immunoblotting, aortic smooth muscle cells were isolated as
above and grown in 35-mm dishes. Cells were washed and collected in
HBSS, and then spun at 3,000 rpm. The cell pellet was resuspended in
lysis buffer (25 mM HEPES, pH 7.5, 1 mM EDTA, 1
mM EGTA, 1 mM dithiothreitol, 0.5 µg/ml
aprotinin, 0.2 µg/ml leupeptin, 1 µg/ml pepstatin A, 42 µg/ml
tosylargininemethylester, 21 µg/ml tosylphenylalaninechloroketone,
133 µM phenylmethanesulfonylfluoride) and the cells were
disrupted by four cycles of freeze thawing. After addition of NaCl to
make the solution 1 M NaCl, the cell slurry was spun at
60,000 rpm in a Beckman Coulter, Inc. ultracentrifuge 20
min at 4 C. The supernatant fraction containing cytosolic proteins was
stored at –70 C until use. The pellet was resuspended in lysis buffer
containing 100 mM NaCl and 1% cholate and extracted on ice
for 30 min. After another centrifugation at 60,000 rpm at 4 C, the
supernatant fraction containing membrane proteins was frozen at –70 C
until use.
For immunoblotting, pituitaries were collected from 10–40 animals and rapidly frozen in DMEM with 10% FBS and 10% dimethylsulfoxide in liquid nitrogen, then stored at –70 C until use. Pituitaries were then thawed and minced finely with a sterile razor blade in a small volume of homogenization buffer (10 mM Tris, pH 7.4, 5 mM EDTA, 1 mM dithiothreitol, and protease inhibitors as above) and homogenized in a Dounce homogenizer at 4 C. The slurry was spun at 17,000 x g for 15 min at 4 C, and the pellet was resuspended in two volumes of homogenization buffer containing 1% cholate. After 1 h at 4 C with occasional mixing, the tube was centrifuged as above and the supernatant fraction was used for immunoblotting. SDS polyacrylamide gels (9 or 12% polyacrylamide) were run and proteins were electrophoretically transferred to nitrocellulose membranes. For Western blotting, primary antibodies were used at the concentrations indicated; horseradish peroxidase-conjugated secondary antibody was used at 1:2,000–1:5,000 dilution and proteins were visualized with enhanced chemiluminescence.
Immunofluorescence Microscopy
Immunocytochemistry was performed essentially as described (52).
All steps were carried out at room temperature. Cells grown 24–48 h on
glass coverslips were treated as indicated in experiments, rinsed three
times in PBS, and fixed in a 4% paraformaldehyde solution in PBS for
30 min. After three washes with PBS, cells were permeabilized in
blocking buffer containing 0.2% Nonidet P-40 and 5% goat serum in
serum-free Ham’s F10 medium. Coverslips were incubated for 60–180 min
in primary antibody diluted as indicated in blocking buffer. Cells were
washed four times for 5 min with PBS and then incubated for 30 min with
fluorescently tagged secondary antibody at 1:100 dilution in blocking
buffer. Images were captured using Metamorph software.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
This work was supported by NIH Grant DK-19974 (P.M.H.) and Mentored Clinical Scientist Award DK-02439 (T.K.G.).
Received for publication August 7, 2000. Revision received October 6, 2000. Accepted for publication October 13, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and ß subunits. J Biol Chem 268:9667–9674 subunit of Gq.
J Biol Chem 269:25335–25338 subunits. J Biol Chem 268:4573–4576 subunits. FEBS Lett 315:340–342[CrossRef][Medline]
-subunits. Nature 360:684–686[CrossRef][Medline]
and ß subunits of trimeric GTP-binding protein. Proc
Natl Acad Sci USA 90:5297–5301- and ß -subunits. Recent insights from studies of
the phospholipase c-ß isoenzymes. Biochem Pharmacol 54:429–435[CrossRef][Medline]
and inhibition of adenylyl cyclase by
Gi1 and Go. J Biol Chem 271:23458–23463 and ß subunits of Gi3. Mol Pharmacol 47:1172–1179[Abstract]
2) of phospholipase C family in mature rat
brain. Neurosci Lett 182:17–20[CrossRef][Medline]
i2-deficient mice. Nat Genet 10:143–150[Medline]
o1 generates Go3, an abundant
G protein in brain. Proc Natl Acad Sci USA 96:1327–1332q. Proc
Natl Acad Sci USA 94:751–756 in chemoattractant-mediated signal
transduction. Science 287:1046–1049This article has been cited by other articles:
 |
|
 |
|
  K. Jian, R. Barhoumi, M. L. Ko, and G. Y.-P. Ko Inhibitory Effect of Somatostatin-14 on L-Type Voltage-Gated Calcium Channels in Cultured Cone Photoreceptors Requires Intracellular Calcium J Neurophysiol, September 1, 2009; 102(3): 1801 - 1810. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Filis, T. Lannagan, A. Thomson, A. A. Murray, P. C. Kind, and N. Spears Phospholipase C-{beta}1 Signaling Affects Reproductive Behavior, Ovulation, and Implantation Endocrinology, July 1, 2009; 150(7): 3259 - 3266. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Liu, M. S. Ruiz, D. A. Austin, and N. J. G. Webster Constitutively Active Gq Impairs Gonadotropin-Releasing Hormone-Induced Intracellular Signaling and Luteinizing Hormone Secretion in L{beta}T2 Cells Mol. Endocrinol., August 1, 2005; 19(8): 2074 - 2085. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Goubaeva, M. Ghosh, S. Malik, J. Yang, P. M. Hinkle, K. K. Griendling, R. R. Neubig, and A. V. Smrcka Stimulation of Cellular Signaling and G Protein Subunit Dissociation by G Protein {beta}{gamma} Subunit-binding Peptides J. Biol. Chem., May 23, 2003; 278(22): 19634 - 19641. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Liu, I. Usui, L. G. Evans, D. A. Austin, P. L. Mellon, J. M. Olefsky, and N. J. G. Webster Involvement of Both Gq/11 and Gs Proteins in Gonadotropin-releasing Hormone Receptor-mediated Signaling in Lbeta T2 Cells J. Biol. Chem., August 23, 2002; 277(35): 32099 - 32108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. R. Albert Editorial: Dopamine-D2-Mediated Inhibition of TRH-Induced PLC Activation in Pituitary Cells--Direct or Indirect? Endocrinology, March 1, 2002; 143(3): 744 - 746. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
